Recruitment and dissociation of NHEJ proteins at a DNA double-strand break in Saccharomyces cerevisiae

Nonhomologous end joining (NHEJ) is an important DNA double strand break (DSB) repair pathway that requires three protein complexes in Saccharomyces cerevisiae, the Ku heterodimer (Yku70-Yku80), MRX (Mre11-Rad50-Xrs2) and DNA ligase IV (Dnl4-Lif1), as well as the ligase-associated protein Nej1. Here we use chromatin immunoprecipitation from yeast to dissect the recruitment and release of these protein complexes at HO endonuclease-induced DSBs undergoing productive NHEJ. Results revealed that Ku and MRX assembled at a DSB independently and rapidly after DSB formation. Ligase IV appeared at the DSB later than Ku and MRX and in a strongly Ku-dependent manner. Ligase binding was extensive but slightly delayed in rad50 yeast. Ligase IV binding occurred independently of Nej1, but instead promoted loading of Nej1. Interestingly, dissociation of Ku and ligase from unrepaired DSBs depended on the presence of an intact MRX complex and ATP binding by Rad50, suggesting a possible role of MRX in terminating a NHEJ repair phase. This activity correlated with extended DSB resection, but limited degradation of DSB ends occurred even in MRX mutants with persistently bound Ku. These findings reveal the in vivo assembly of the NHEJ repair complex and shed light on the mechanisms controlling DSB repair pathway utilization.

Key Words: chromatin immunoprecipitation, double strand break, nonhomologous end joining, resection, yeast

This article has been cited by other articles:

				P. L. Palmbos, D. Wu, J. M. Daley, and T. E. Wilson Recruitment of Saccharomyces cerevisiae Dnl4-Lif1 Complex to a Double-Strand Break Requires Interactions With Yku80 and the Xrs2 FHA Domain Genetics, December 1, 2008; 180(4): 1809 - 1819. [Abstract] [Full Text] [PDF]

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